FIGURE 7. A peptide inhibiting association of MMP-9 andβ1-integrin reduces thrombin-mediated cellular invasion.

Panel A, protein binding assay. The MMP-9 hemopexin domain was coated to ELISA plates and incubated with an α5β1-integrin-Fc fusion protein. Bound protein was detected via the Fc part of the fusion protein using horseradish peroxidase-conjugated protein A. The values for the protein detected on coated SA as negative control were subtracted from the corresponding MMP-9-bound signals. α5β1-integrin binds to the MMP-9 hemopexin domain in a dose-dependent manner. Panel B, immunoprecipitation, U2-OS cells were preincubated with or without a peptide spanning the LRSG sequence in the hemopexin domain of MMP-9, a scrambled control peptide, or a MMP-9 antibody raised against a sequence in the hemopexin domain adjacent to LRSG followed by stimulation with thrombin. Cell lysates (250 µg) were immunoprecipitated with a β1-integrin antibody and immunoblotted with a MMP-9 antibody raised against the hemopexin domain. Panel C, gelatinase assay. U2-OS cells were seeded in a 96-well plate, cultured in serum-free media, and stimulated with thrombin, or with thrombin and treated with the LRSG-peptide. uenched gelatin was added and fluorescence measured at the indicated times as described in the legend to Fig. 2E. Panels D–F, invasion assays. U2-OS cells were plated on a Matrigel-coated modified Boyden chamber and then incubated with or without thrombin, the LRSG, or a scrambled control peptide (D) or panel E using the antibody against the MMP-9 hemopexin domain. Panel F, TE-85 osteosarcoma cells were plated on a Matrigel-coated modified Boyden chamber and then incubated with or without 4 units/ml of thrombin. Invasion assays (D–F) were performed in triplicate with a minimum of 10 grids (40× magnification) per filter counted (n.s., not significant, * indicates p < 0.05, *** indicates p < 0.001). All experiments were repeated at least three times.